On-stage selection of single round spermatids using a vital, mitochondrion-specific fluorescent probe MitoTracker (TM) and high resolution differential interference contrast microscopy

The selection of individual round spermatids for round spermatid injection (ROSI), a prerequisite for the successful application of this infertility t reatment, has been hampered by the ambiguous definition of a round spermati d and the lack of specific vital and non-vital markers. Using cells from rh esus monkey and bull, we describe a non-invasive method for the on-stage se lection of individual round spermatids for ROSI, based on the polarized pat terns of mitochondria, visualized in live round spermatid cells by epifluor escence microscopy after incubation with MitoTracker(TM), a vital, mitochon drion-specific fluorescent probe. The correct identification of live round spermatid was confirmed by the presence of the acrosomal granule or acrosom al cap in parallel observations by Nomarski differential interference contr ast microscopy. The existence of mitochondrial polarization was first estab lished by the labelling of MitoTracker-tagged round spermatids with spermat id-specific antibodies against proteins of nascent sperm accessory structur es combined with antibodies against a nuclear pore complex component, known to disappear at the round spermatid stage. Using an inverted microscope eq uipped with epifluorescence, the round spermatids can be individually selec ted from a heterogeneous population of testicular cells labelled with MitoT racker dyes, A major advantage of this approach is that the dyes are incorp orated into the paternal mitochondria, destined for rapid elimination after fertilization, In addition, the relatively high excitation and emission wa velengths of MitoTracker dyes are less harmful to DNA after their photon ex citation, Before the appropriate clinical testing is conducted, the MitoTra cker-based round spermatid selection may be instrumental in the training of clinical staff.